We are developing a novel cytoprotective small molecule for the prevention of limb ischemia-reperfusion injury (LIRI). Acute lower extremity ischemia remains a significant problem with a mortality rate ranging from 9-42% in elderly patients and a 20% amputation rate in survivors. Despite such significant morbidity and mortality, therapeutic strategies have not advanced over the past two decades, with the administration of fluid resuscitation, thrombolysis and anticoagulant therapy remaining as the mainstays of treatment. LIRI is produced by an acute alteration in the synthesis of free radicals, wherein a) nitric oxide (NO) is profoundly depleted, due to the arginase-1 mediated consumption of L- arginine, the substrate for endothelial NO synthase (ecNOS), and b) superoxide anion rises to toxic levels, produced by uncoupled ecNOS and hyperactivation of xanthine and NAHDP oxidases. The imbalance of these two free radical species damages muscle directly, via the production of peroxynitrite and its activation of poly(ADP-ribose) polymerase (PARP), and indirectly, via an effect on the microcirculation by inducing vasoconstriction (and thus further ischemia), platelet adhesion and aggregation, and neutrophil adhesion and degranulation. Replenishment of NO and removal of superoxide are each partially effective, but greater protection of muscle injury is expected from the simultaneous correction of both abnormalities. To address this unmet clinical need, Radikal Therapeutics is developing R-100, a novel agent formed from the covalent linkage of an organic nitrovasodilator that releases NO, and a pyrrolidine nitroxide that acts as a superoxide dismutase mimetic, catalase mimic, and peroxynitrite decomposition catalyst. In combination, these functionalities allow R-100 to remove toxic reactive oxygen species and deliver NO without the confounding effect of producing peroxynitrite. Preliminary data reveal that R-100 is: 1) a potent vasodilator in rat and ovine hypertension models;and 2) a profound tissue-protective agent in a murine model of circulatory shock. Phase 1 Specific Aim: Establish the dose-dependent efficacy of R-100 in preventing tissue injury in an experimental murine model of LIRI. In conjunction with Professor Michael Watkins (Harvard Medical School), we will carry out a randomized, single-blinded, placebo-controlled investigation of R-100 in a murine model of LIRI. Anesthetized C57BL6 mice will be subjected to 1.5 h of unilateral limb ischemia, followed by 24 h of reperfusion. A sham injury group will be compared to treatment with R-100 (0, 20, 40, and 80 mg/kg/day) 10 min before the onset of reperfusion. After 24 h of reperfusion, mice will be euthanized, limbs harvested, and the posterior hind-limb muscle placed in 10% paraformaldehyde for histologic examination or snap-frozen in liquid N2 for biochemical analysis. We expect that R-100 will dose-dependently reduce tissue injury, as reflected in reductions in lipid peroxidation, neutrophil infiltration, rhabdomyolysis, poly(ADP-ribose) formation, and gastrocnemius ATP concentration. These treatment effects are expected to translate into clinical endpoints in limb reperfusion injury of: 1) increased limb viability, 2) diminished rhabdomyolysis, 3) shorter hospitalization, and 4) reduction in all-cause 30 day mortality. PUBLIC HEALTH RELEVANCE: Loss of blood flow to a limb is a medical emergency requiring immediate restoration of perfusion. Therapies that reopen blocked vessels may paradoxically induce further tissue damage, known as "reperfusion injury". There are no approved therapies to prevent or treat this condition. We are developing a novel drug that targets the basic mechanisms of reperfusion injury and is intended as a prophylactic agent in patients undergoing revascularization to restore blood flow to the limb. We will test this agent in a clinically-relevant small animal model of acute limb ischemia.